The present invention relates generally to analogs of endogenous glycoproteins, and particularly to analogs of secreted proteins capable of inducing hematopoetic cell development.
Many proteins secreted by cells into the extracellular environment acquire covalently attached carbohydrate units following translation, typically in the form of oligosaccharide units linked to asparagine side chains by N-glycosidic bonds. Both the structure and number of oligosaccharide units attached to a particular secreted protein can be highly variable, resulting in a wide range of apparent molecular masses attributable to a single glycoprotein. Many regulatory proteins having potential utility as human and veterinary therapeutic agents are secreted glycoproteins of this type. Attempts to express such proteins in recombinant systems have been complicated by the heterogeneity attributable to this variable carbohydrate component.
Immunoregulatory proteins, or lymphokines, are a class of secreted proteins; many are glycoproteins. For example, growth and differentiation of hematopoeitic cells is mediated by a number of glycoproteins collectively referred to as colony stimulating factors or CSFs. In humans, these proteins include granulocyte-macrophage colony stimulating factor (GM-CSF), a glycoprotein required for the production of granulocytes and macrophages from normal bone marrow which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Other human CSFs (hCSFs) include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, granulocyte CSF (G-CSF), which stimulates development of granulocytes, and burst promoting activity (BPA), which induces development of erythroid cell progenitors into hemoglobin-containing cells. An additional murine CSF, known variously as IL-3 or multi-CSF, stimulates the development of numerous cell types of hematopoeitic lineage. A human homologue of murine IL-3 has recently been reported.
GM-CSF was initially identified as a 23 kilodalton protein present in preparations obtained from endotoxin-conditioned mouse lung. See Burgess et al., J. Biol. Chem. 252:1998 (1977). Human GM-CSF activity was partially purified from placental conditioned medium by Nicola et al., Blood 54:614 (1979). Human GM-CSF has also been identified in cultures of the human T-lymphoblast cell line Mo, and shown to modulate the activities of mature neutrophilic granulocytes by Gasson et al., Science 226:1339 (1984). Cloning and expression of recombinant human GM-CSF from various sources has been reported by Cantrell et al., Proc. Natl. Acad. Sci. U.S.A. 82:6250 (1985); Wong et al., Science 228:810 (1985); and Lee et al., Proc. Natl. Acad. Sci. U.S.A. 82:4360 (1985). Cantrell et al. isolated human GM-CSF sequences from cDNA libraries prepared from the HUT-102 cell line. The isolated human sequences were shown to direct synthesis of a biologically active GM-CSF using a yeast expression system.
Recombinant proteins which are expressed, glycosylated and secreted by yeast typically contain variable quantities of associated carbohydrate. Thus, purified mixtures of recombinant glycoproteins such as human or murine GM-CSF can consist of from 0 to 50% carbohydrate by weight. Miyajima et al., EMBO Journal 5:1193 (1986) reported expression of a recombinant murine GM-CSF in which N-glycosylation sites had been mutated to preclude glycosylation and reduce heterogeneity of the yeast-expressed product.
The presence of variable quantities of associated carbohydrate in recombinant secreted glycoproteins complicates purification procedures, thereby reducing yield. In addition, should the glycoprotein be employed as a therapeutic agent, a possibility exists that recipients will develop allergic reactions to the yeast carbohydrate moities, requiring therapy to be discontinued. For these reasons, biologically active, homogeneous analogs of immunoregulatory glycoproteins having reduced carbohydrate are desirable for therapeutic use.
It has now been found that functional mutant analogs of human GM-CSF and other human CSFs normally secreted as glycoproteins can be produced by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in higher yields than wild-type homologues, using yeast expression systems. The reduced-carbohydrate analogs of human GM-CSF exhibit a specific activity equivalent to comparable purified mixtures of glycosylated recombinant human GM-CSF in appropriate assays of colony-stimulating activity.